The phenotypic makeup and functional capacity of murine and human hematopoietic stem cells (HSC) has been exhaustively investigated making HSC one of the most-characterized groups of cells in mammalian biology. Still, not a single functional marker has been previously identified that is common to both murine and human HSC while being also expressed on at least three important cellular components of the hematopoietic niche (HN), namely osteoblasts (OB), endothelia cells (EC), and mesenchymal stem cells (MSC). Our focus on CD166 began when we first established that CD166 identifies OB that mediate a hematopoiesis enhancing activity. We also published that CD166 is expressed on normal murine and human HSC and plays a key role in determining their functional capacity and the competence of the HN. We recently confirmed that CD166 is expressed on a subset of EC and nestin+ MSC. Since CD166 is capable of mediating hetero- and homophilic cell-cell interactions, we propose that CD166 serves as both ligand and receptor of HSC interactions with cellular elements of the niche and is an important regulator of the competence of the HN and of stem cell function. HSC from CD166-/- mice engraft poorly in lethally irradiated hosts. On the other hand CD166-/- mice do not support the engraftment of LT-HSC suggesting that loss of CD166 abrogates the competence of the HN. Marrow-homed LSK cells from wildtype (WT) donors lodged closer to the endosteum of host mice than similar cells from knockout (KO) animals suggesting that engraftment is adversely impacted by the lack of HSC-OB homophilic CD166-interactions7. Single-cell RNAseq analysis of WT and CD166-/- CD150+CD48- LSK cells incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activated cytokine signaling, epigenetic pathways, stem cell pluripotency genes, and mitochondria-related signaling pathways. Based on these findings, we hypothesize that CD166 is a functional marker required for HSC- niche interactions that support the competence of the hematopoietic niche. We also believe that preferential CD166 homophilic interactions in the niche contribute to HSC regulation and maintenance of the hematopoietic potential of stem cells. We will test these hypotheses by investigating 2 aims: 1) Test the hypothesis that CD166 expression on OB, EC, and MSC in the hematopoietic niche is required for the competence of the hematopoietic niche and 2) Test the hypothesis that hematopoietic stem cells lacking CD166 homophilic interactions with OB, EC, and MSC in the niche fail to develop normally and acquire an engraftment defect. The significance of this proposal is that it will examine the role of a new and unique functional marker of human and murine HSC and will investigate the impact of CD166 modulation on stem cell function. The novelty of this proposal stems from its potential to implicate CD166 in the regulation of the HN and HSC that reside in and interact with CD166-rich cellular elements of this microenvironment. These studies have the potential to define how homophilic CD166 interactions promote HSC self-renewal.